Nanostructured Biomimetic  Superconductive Devices of Making and Its Multiple Applications Thereto

ABSTRACT

A multiple functioning superconductive device was invented based on Toroidal Josephson Junction (FFTJJ) array with 3D-cage structure self-assembled organo-metallic superlattice membrane. The device not only mimics the structure and function of an activated Matrix Metalloproteinase-2 (MMP-2) protein, but also mimics the cylinder structure of the Heat Shock Protein (HSP60) protein, that works at room temperature under a normal atmosphere, and without external electromagnetic power applied. The device enabled direct rapid real-time monitoring atto-molarity concentration ATP in biological specimens and was able to define the anti-inflammatory and pro-inflammatory status revealed a transitional range of ATP concentration under antibody-free, tracer-free and label-free conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. Non-Provisional Patent Application inthe title of Nanostructured Biomimetic Superconductive Devices of Makingand Its Multiple Applications Thereto, that claims the benefit of theU.S. provisional patent application No. 62/848,422, in the title ofNanostructured Biomimetic Organo-metallic Superconductive Devices ofMaking and its applications for Direct Real-time MonitoringAtto-Molarity ATP in Biological Specimens” filed on May 15, 2019. Theentire disclosure of the prior Patent Application Ser. No. 62/848,422 ishereby incorporated by reference, as is set forth herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of applications insuperconductor, in particular, to a device having both characteristicsin superconductivity and memristive/memcapacitive/meminductive embeddedwith non-ferromagnetic switches functioning at room-temperature and itsapplications in sensing biological specimens.

BACKGROUND OF THE INVENTION

Real-time monitoring of ATP in biological specimens is a convenientmeans to monitor the hygiene in healthcare units and for medicaldevices. Rapid and precise monitoring has been in strong demand from thepublic and under tight regulations [1-8]. Researchers found a rapidmeasurement of ATP in series diluted bacterial cultures correlates withthe bacterial concentrations contaminated in the biological specimens,thus ATP testing becomes an important indicator for monitoring thehygiene in healthcare units [9-11].

The predominate luciferase bioluminescent sensor method, thefluorescence in situ hybridization (FISH) method, the HPLC method, theflow cytometry method and the immobilized gene electrochemical sensormethod are common methods for ATP [7-14]. Among these methods, theluciferase bioluminescent sensor method has been recommended by CDC forassessing hospital device surface hygiene and has been used for decades.Nante's review article revealed the ATP luciferase bioluminescentportable sensor method is not a standardized methodology, because eachtool has different benchmark values, not always clearly defined. Theauthors stressed this technique could be used to assess, in real time,hospital surfaces cleanliness, but has its limitations of not accuratein detecting bacteria and the requirement of the washing out of thedisinfectant step on the surfaces before testing are drawbacks [14]. Thegene method could improve the detection limits to 10 fM ATP, but it isnot real-time monitoring and the procedures are burdensome [12]. EvenNASA recent recognized an unmet need for real-time on-line assessing thedrinking water contamination for astronauts who are in their spaceflight, because all current methods used for monitoring water qualityincluding the ATP bioluminescent and gene methods were deemed unfit,because the requirements of (1) rapid accurate reagent-free real-timetesting; (2) the testing machine needs to be lightweight and small insize; (3) there should be no sample preparation steps, have not met[15]. In the face of the demand and challenges, our group proposed aninnovative approach to attack the problems, that is to developsuperlattice nanostructured superconductive/memristive sensors havingorgano-metallic crossed-linking polymer membranes which work atJosephson Junction at the zero-bias potential, may overcome proteinnonspecific bounding and increase accuracy, based upon our priorexperience using the superconductive/memristive device to enabledirectly detection of collagen-1 in 16.7 atto molarities with higherthan 96% accuracy using fresh human capillary blood serum specimens atlow (8.3 fM) and high (0.55 nM) levels having imprecision of 4.9% and0.8%, respectively compared with the controls, under antibody-free,tracer-free, and reagent-free conditions at room temperature [16-17].

Recent theoretical predictions of Josephson-based meminductive andmemristive quantum superconducting devices not only have multiple statesuperposition property, but also the Cooper-pair waves behavehysteretically have drawn attention [18-20]. Herein by utilizing theseproperties, we assert that significantly improving ATP testing methodmay be accomplishable. We planned to develop two types of sensors,namely, the biomimetic matrix metalloproteinase (MMP-2) Sensor 1 at itsactive state by a heating method to switch “Off” the cysteine group inthe membrane, which is ready to biocommunication with ATP; then theresults due to ATP's interaction will be compared with Sensor 2, also atits active state of the biomimetic MMP-2, but by a direct fabricationmethod of organo-metallic self-assembled cross-linked polymer withoutcysteine. It is well known that superlattice membranes have been used ascandidates for applications in superconductivity [21].

Literature reported ATP hydrolyzation extracellularly induces cancercells drug resistance [22-25]. Assessing human milk immunologicaladvantage over cow milk in preventing extracellular ATP hydrolyzation isimportant to human health. Shonhai's group published a review articleregarding the roles of the Heat Shock Proteins (HSP) acted asimmunomodulates whose capability is to transform the anti-inflammatoryproperty when the HSP concentrations are low to the pro-inflammatoryproperty when HSP concentrations are high [26], and unfortunately theauthors did not define the concentration range. We felt an unmet needexists for this health-related important topic. Extracellular ATPconcentration and intracellular ATP concentration ranges are importantrelated to HSPs' functions either in physiological or pathologicalfunction, because HSPs primarily occur extracellularly, but alsoreported from literature, HSPs occur in an intracellularmicro-environment [27]. If we can build a well-characterized andwell-controlled system to define the critical HSP concentration rangesin the transformation between the two statuses that would be a primaryattempt toward a resolution.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new generation oforgano-metallic superconductive, memristive/memcapacitive devicecompromising of the active sites of a biomimetic MatrixMetalloproteinase (MMP-2) in the membrane of the device.

It is an object of the present invention to provide a new generation oforgano-metallic superconductor/memristor/memcapacitor withmultiple-layer curvature structure mimicking the function and structureof cation-diffusive facilitator (CDF) protein YiiP of E. Coli(gram-negative), and to efflux zinc ions from cellular membrane.

It is an object of the present invention to further provide thebiomimetic YiiP protien which is capable to be a periplasmic zincchaperone for providing zinc to the E. Coli when it is in a starvationof zinc state.

It is a further object of the present invention to provide thebiomimetic YiiP protien which is capable to be a Heat Shock Protein(HSP60) chaperone of GroEL in helping folding of a protein in itscavity.

It is a further object of the present invention to provide thebiomimetic MMP-2 sensor which is able to sense aM ATP concentrationpresence in biological samples in a direct, rapid real-time monitoringfashion of hospital instrumental hygiene without using antibody,labeling and other burdensome procedures.

It is a further object of the present invention to provide thebiomimetic MMP-2 sensor which is able to be a memory storage and asuperlattice quantum computing chip working at room temperature.

It is a further object of the present invention to provide a methodwhich can be optimally operated for fabrication of the self-assembled3D-nanocage structure multiple-layer membrane on an electrode surfaceacted as a living biological cell model to assess the immunomodulantconcentration effect.

It is a further object of the present invention to provide a devicehaving multiple utilities, not only in sensing of multiple analytes, butalso can be used for defining the immunomodulant concentration effect ontransformations from an anti-inflammatory to a pro-inflammatory statusin the presence of LPS challenge with a wide range ATP concentrationchanges in the biological specimens.

It is a further object of the present invention to provide a device todirect monitor the reversible membrane potential change under theinfluence of ATP concentrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the 2D AFM membrane image in the native state of thebiomimetic MMP-2 device. FIG. 1B depicts the cross-section analysis ofthe AFM membrane image.

FIG. 2A depicts the 3D AFM image of the activated biomimetic MMP-2Sensor 1 by the heating method. FIG. 2B depicts the enlarged 2D AFMimage with the same z value range between 33.2 to −21.4 nm as the nativestate device in FIG. 1A for comparison.

FIG. 3A depicts the 2D image of the multiple-layer ring structure of theactivated biomimetic MMP-2 Sensor 2 by the direct fabrication method in0.734×0.734 μm². The ring structure parameters labeled as “1” arelisted. FIG. 3B depicts the cross-section analysis of the AFM membraneimage. FIG. 3C depicts the 3D AFM image from the bird-view. FIG. 3Ddepicts the enlarged image in 0.9×0.9 μm² shown in the flat area in FIG.3I. FIG. 3E depicts the alternative band deep-band flat formation of themembrane which promotes the Cooper-pair electron cloud mobility in1.6×1.6 μm². FIG. 3F depicts the enlarged 2D AFM image at high sensormode in a top view of the biomimetic Heat Shock Protein (HSP) 60chaperone structure having 7 subunits as shown on the top. The arrowindicates the mobile zinc ions. FIG. 3G depicts the top view in anamplitude mode of the AFM image of the large single porous biomimeticHSP60 structure. FIG. 3H depicts the side view of the 3D AFM image inhigh sensor mode for the single porous structured in 3×3.6 μm². FIG. 3Idepicts the 3D AFM image of the tall cylinder structure of thebiomimetic HSP60 in 10×10 μm².

FIG. 4 illustrates the scheme of the steps of direct detection of ATPusing the 3D-nanocage structure biomimetic MMP-2 membranesensor/superconductor approach. Step 1: the Direct Electron-relay (DER)model of the 3D-nanocage polymer network structure between the zinc ionsfrom the activated biomimetic MMP-2, by elimination of the cysteinegroup from the polymer mixture of dm-β-DMCD/TCD/PEG/PVP/ZnCl₂ beforedeposited on the gold chip, and the mm-β-DMCD (short names as MCD) intris/HCl buffer. Step 2: Form a long range DET in the presence ofTris/MCD media with ATP being included in the cyclodextrin cavities,that not only stabilized ATP, but also invited ATP participated in thelong range direct electron-relay chain between the media and the3D-nanocage membrane electrode assembling (MEA); Step 3: the ATPinteracts with the cage polymer network molecules forming enhanced DERwithout a need for an external power supply.

FIG. 5A depicts activated Sensor 1 by the heating method's i-V curves indifferent scan rate from 1 Hz to 25 kHz in pH 7.8 buffer (20 mM Tris/HClbuffer with 3 mM KCl, 130 mM NaCl and 0.5 μg/mL MCD), in short, as theTris/HCl/MCD buffer. FIG. 5B depicts Sensor 1's i-V curves under theinfluence of ATP concentrations over 40 fM to 60 nM compared with thecontrol under the scan rate 60 Hz in the Tris media.

FIG. 6 depicts the scan rate impacts on the i-V curves of the activatedstate biomimetic MMP-2 Sensor 2, by the direct fabrication method, overscan rate from 1 Hz to 25 kHz in the tris/HCl/MCD media.

FIG. 7 depicts Sensor 2's i-V curves transformation between memristiveto superconductive at zero-bias potential under the influence of ATPfrom 25 aM to 2 μM compared with the control with a 60 Hz scan rate inthe tris/HCl/MCD media. The Panel A is the i-V curves for ATP 25 aMcompared with the control; the Panel B is for the i-V curve having 400aM; the Panel C: ATP 400 fM; the Panel D: ATP 200 pM; The Panel E: ATP200 nM; the Panel F: ATP 2 μM.

FIG. 8 depicts the comparison of the superconducting i-V curves at thezero-bias potential for Sensor 2 under the influence of ATP from 200 fMto 200 μM compared with the control under a 10 kHz scan rate in thetris/HCl/MCD media. The Panel A: is the control; the Panel B: ATP 200fM; the Panel C: ATP 400 fM; the Panel D: ATP 200 pM; the Panel E: ATP200 nM; the Panel F: ATP 400 nM; the Panel G: ATP 800 nM; the Panel H:ATP 2.0 μM, and the Panel I: ATP 200 μM in the media.

FIG. 9 depicts the supercurrent vs. ATP concentration curves of theDET_(red) and DET_(ox) peaks at the zero-bias potential with a 60 Hzscan rate for Sensor 2. The ATP concentration ranges are from 400 aM to2 μM. Data obtained according to Figures from FIG. 7B to 7F.

FIG. 10 depicts the supercurrent vs. ATP concentration curves of theDET_(red) and DET_(ox) peaks at the zero-bias potential at a 10 kHz scanrate for Sensor 2. The ATP concentration ranges are from 200 fM to 200μM. Data obtained according to FIGS. 8A to 8I.

FIG. 11 depicts the impact of ATP concentrations at the Josephsonjunction on the supercurrent and the impact on the phase change of thewaves at the zero-bias potential during 10 consecutive scans at 60 Hzover the ATP concentrations from 400 fM (the Panel A), 200 pM (the PanelB), 200 nM (the Paneol C), and to 2.0 μM (the Panel D) in theTris/HCl/MCD media.

FIG. 12A depicts 25 aM low concentration ATP in consecutive 5 scancycles of san rate 60 Hz i-V curves compared with the control of Sensor2. There was no phase change occur, and keeping the hysteresis curves.FIG. 12B depicts 400 fM concentrations ATP in consecutive 9 scan cyclesof san rate 60 Hz i-V curves compared with the control, having a phasechange occur and superconducting at zero-bias potential. FIG. 12C alsodepicts in the presence of 200 nM ATP concentrations, Sensor 2 shows thephase change and superconducting current at 60 Hz san rate.

FIG. 13 depicts the 3D dynamic map of the relationships over 5 level ATPconcentrations from 0.2 pM to 2.0 μM over the potential range between−40 mV to +40 mV for illustration of the relationship among ATPconcentrations, zero-bias potential and the differential quantumconductance using 10 kHz scan rate forwarding scan data.

FIG. 14 depicts Sensor 1's real-time direct monitoring of current vs.time profiles over ATP concentrations over 100 aM to 400 nM (9 levelsfrom curve a to k) vs. controls in the buffer solution. Samples runtriplicates.

FIG. 15 depicts the lower end concentration curves from a (the control)to e. Inserts are enlarged view for low end concentration compared withcontrols.

FIG. 16 Panel A depicts the calibration curve of current density aftersubtracted the background current vs. ATP over 100 aM to 170 nM. FIG. 16Panel B depicts the calibration curve over the linear range from 400 fMto 170 nM. Each sample run triplicates.

FIG. 17 depicts Sensor 2's open circuit potential curves for monitoringthe energy change over 25 aM-400 pM ATP.

FIG. 18 depicts the calibration curve of voltage vs. ATP concentrationsover 25 aM-400 pM.

FIG. 19 depicts Sensor 2's open circuit potential curves at the high endATP concentration over 0.8 nM to 2.0 μM.

FIG. 20 depicts the double log plot of the calibration curve of the opencircuit potential vs. ATP concentrations from 0.8 nM to 2.0 μM.

FIG. 21 depicts Sensor 2's voltage vs. time curves by the Double-stepChronopotentialmetry (DSCPO) method in the presence of various ATPconcentrations from 25 aM to 400 nM in the Tris/HCl/MCD buffer comparedwith the control. Each sample run triplicates.

FIG. 22A depicts the calibration curve of the normalized actionpotential divided by the mean signal vs. ATP concentrations over 100 aMto 200 nM. FIG. 22B depicts the low end ATP's calibration curve from ATP25 aM to 400 aM in Action potential of DER peak.

FIG. 22C depicts the semi-log plot of the absolute normalized restingpotential vs. ATP concentrations under the same experimental conditionsas FIG. 22A.

Table 1 shows a comparison of method performance for quantitation of ATPspiked in the milk samples using the voltage method

FIG. 23 depicts the Anti-inflammatory and the Pro-inflammatory countermap related to the ATP concentration at zero-bias, and the quantumconductance for the “Healthy” HSP60 Sensor 2.

FIG. 24 shows the semi-log plot curve of ATP concentration ranges effecton the cell Reversed Membrane Potential (RMP) (after subtracting thecontrol). It was shown the trace is not depending upon the ATPconcentrations between the range from 100 aM to 800 nM with n=24, eightconcentration levels.

FIG. 25 depicts the semi-log plot curve of ATP concentration rangeseffect on the ratio of action potential vs. resting membrane potential.

FIG. 26 Left Panel depicts the organic milk CV curves for current vs.applied potential in the presence of 60 nM ATP compared with the milkcontrol (in black color) in 6 consecutive scans at 60 Hz. FIG. 26 RightPanel depicts the CV curves of with 60 nM ATP (in red color) in buffermedia and curves of the buffer control.

FIG. 27 depicts the DET peaks' current vs. scan cycles: top Panel is forthe DET_(red) peak and the bottom Panel is for the DET_(ox) peak.

FIG. 28A depicts the first scan cycle at 60 Hz in the presence of 60 nMATP (final concentration) in human milk sample compared with the milkcontrol sample. FIG. 28B depicts the second scan cycle at 60 Hz in thepresence of 60 nM ATP (final concentration) in human milk samplecompared with the control milk sample. FIG. 28C depicts the third scancycle. FIG. 28D depicts the fourth scan cycle.

FIG. 29 depicts the alternative trends of the superconducting current atthe zero-bias vs. scan cycles for the forward scan and backward scancompared with the human milk controls, respectively.

FIG. 30 depicts the overlapping curves of the control human milk samplein 6 consecutive scans vs. the milk sample with 60 nM ATP at 60 Hz.

EXAMPLE 1 Fabrication of the Membranes

Sensor 1 has an activated biomimetic MMP-2 membrane by a heating methodat 80° C. for 5 minutes using the innate biomimetic MMP-2 membranefabricated based on a published procedure [34]. Sensor 2 was also in astate of activation of biomimetic MMP-2 by a direct deposited methodwith compositions of TCD, PEG, PVP, bM-β-DMCD and embedded zinc chlorideon gold chips with appropriate proportions at 37° C. for 96 hours. Themorphology of the AU/SAM was characterized using an Atomic ForceMicroscope (AFM) (model Dimension Edge AFM, Bruker, MA).

EXAMPLE 2 The Friedel-Oscillation in the Superlattice Membranes

Friedel-oscillation is a phenomenon of long-range indirect interactionsbetween electrons on a superlattice surface [21]. Evaluations of theFriedel-oscillation were conducted based on the AFM images. FIG. 1Arevealed the strong Friedel-oscillation with a flame-like electric cloudsurrounded on the zinc atoms which the “cloud” moves toward the samedirection in the 1.0 μm² area. This is the evidence of the Cooper pairtransmission waves at the Josephson junction. The image was for thebiomimetic “native” MMP-2 membrane, i.e., the membrane comprised oftriacetyl-ß-cyclodextrin (TCD), polyethylene glycol diglycidyl ether(PEG), poly(4-vinyl pyridine) (PVP), bis-substituted imidazoledimethyl-β-cyclodextrin (bM-β-DMCD), cysteine and embedded zinc chloridein appropriate proportions. The native MMP-2 protein has two states: aninnate state with the cysteine “on” and an activated state with thecysteine “off”. FIG. 1A depicts the 2D AFM membrane image in the nativestate of the biomimetic MMP-2 device. FIG. 1B depicts the cross-sectionanalysis of the AFM membrane image. FIG. 1A was with the cysteine “On”in its innate state, and we observed the Cooper pair electrons movingtoward the same direction. FIG. 2A depicts the 3D AFM image of theactivated biomimetic MMP-2 Sensor 1 by the heating method. FIG. 2Bdepicts the enlarged 2D AFM image with the same z value range between33.2 to −21.4 nm as the native state device in FIG. 1A for comparison.Sensor 1 has an activated biomimetic MMP-2 membrane by a heating methodto kick out the cysteine group in the network, as evidence, we did notobserve moving Cooper pairs in FIG. 2A, the labeled square has the sameZ range as shown in FIG. 1A. The matrix of the superlattice was shownpartially damaged shown in FIG. 2B.

FIG. 3A depicts the 2D image of the multiple-layer ring structure of theactivated biomimetic MMP-2 Sensor 2 by the direct fabrication method in0.734×0.734 μm². The ring structure parameters labeled as “1” arelisted. The inner ring diameter is about 161 nm, and the out ringdiameter is 192 nm. Sensor 2 was directly fabricated using the samepolymers and other compositions such as bM-β-DMCD/TCD/PEG/PVP/ZnCl₂,except without cysteine. The Friedel-oscillation was observed in twolocations in FIG. 3A. FIG. 3B depicts the cross-section analysis of theAFM membrane image. FIG. 3C depicts the 3D AFM image from the bird-view.FIG. 3D depicts the enlarged image in 0.9×0.9 μm² shown in the flat areain FIG. 3I. The Friedel-oscillation observed as the moving flamessurrounded the zinc atoms, while the Cooper pairs moved toward the samedirection and the superlattice matrix was very orderly arranged on thesurface of the FIG. 3D. FIG. 3E depicts the alternative arrangementbetween the valley band and the flat band on the membrane which promotesthe Cooper-pair electron cloud mobility in 1.6×1.6 μm². FIG. 3F depictsthe enlarged 2D AFM image at high sensor mode in a top view of thebiomimetic Heat Shock Protein (HSP) 60 chaperone structure having 7subunits as shown on the top. The arrow indicates the mobile zinc ions.FIG. 3G depicts the top view in an amplitude mode of the AFM image ofthe large single porous biomimetic HSP60 structure. FIG. 3H depicts theside view of the 3D AFM image in high sensor mode for the single porousstructured in 3×3.6 μm². FIG. 3I depicts the 3D AFM image of the tallcylinder structure in a size of 1.3-1.5 μm diameter and 491 nm in heightof the biomimetic HSP60 in 10×10 μm².

EXAMPLE 3 Direct Electron Relay (DER) Model Based on a CylinderCage-Like Structured Polymer Network

FIG. 4A illustrates the Direct Electron-relay (DER) art model made by acage structured polymer network between the zinc ions from the activatedbiomimetic MMP-2, by elimination of the cysteine group from the polymermixture of dm-β-DMCD/TCD/PEG/PVP/ZnCl₂before deposited on the gold chip,and the mm-β-DMCD (short names as MCD) in tris/HCl buffer. Zinc ionscoordinated with three imidazole groups (two from the bm-β-DMCD and onefrom the MCD), and either with the COO⁻ of TCD, or OH⁻ group from thecyclodextrin, or from water. Furthermore all other hydrogen bounding,hydrophobic interaction and the π-π interaction between the functiongroups inside the cavity induced the DER force evidenced as the Cooperpair electron freely moving toward a same direction shown in FIG. 3D andthe cylinder cage structure was shown from FIG. 3F to FIG. 3I.

FIG. 4B depicts the art model when ATP interacts with the cylinderpolymer network molecules that form an enhanced DER. This effect hasenabled ATP played a role to transform a memristive/meminductive sensorto a superconductor at a san rate≥60 Hz with the ATP concentrationhigher than 400 aM at zero-bias potential.

EXAMPLE 4 The JJ Characteristics

The hallmarks of the JJ characteristics are (1) at a DC voltage=0, asupercurrent

I _(s) =I _(c) sin(Δφ)   (1)

I_(c) is critical current, Δφ is the phase difference between the wavesof two superconductors, appears at the DC Josephson junction; (2) at afinite DC voltage, the phase of the supercurrent has changed is afunction of time that caused oscillating at the AC Josephson Junction,which is proportional to 2 eV_(DC,) i.e.,

∂φ/∂t=(2e/h)V _(DC)   (2) [28].

Here the Planck constant=h/2π (1.055×10⁻³⁴ Js). Scan frequency affectsi-V curves shown in FIG. 5A for Sensor 1 is different from that ofSensor 2 in FIG. 6, both figures were working in theTris/mono-substituted imidazole dimethyl-β-cyclodextrin (mM-β-DMCD), inshort, MCD control media over 1 Hz to 25 kHz. Sensor 1 demonstrated amemristive behavior with a hysteresis point at zero V and zero currentat 60 Hz, but it had no superconductivity through all the curves, thatindicates the heating procedure damaged the superlattice structureleading to a poor Cooper pair formation. In contrast, Sensor 2demonstrated a perfect memristive behaves at 60 Hz and also showed thesuperconductivity at zero-bias potential over 5 kHz to 25 kHz as shownin FIG. 6. This fact showed as long as having the Cooper pair formation,the superconductivity can be expected. The sine waves oscillated in thehigh scan frequency are observed.

EXAMPLE 5 Superconductivity with Super-Positioning

Because Sensor 1 lacks of Friedel-oscillation due to the heating processfor eliminating the cysteine group, without any superconductivityregardless with or without ATP in the Tris media as shown in FIGS. 5A(without ATP) and 5B (with ATP) over a wide rage of scan rate. Scanfrequency effects on the Shapiro step voltage in the sine waves ofSensor 2 over 5 kHz to 25 kHz, under external magnetic field=0condition, was observed as shown in the i-V curves in FIG. 6, FIG. 7 andFIG. 8 for without and with ATP under 60 and 10 kHz scan rate,respectively. The superlattice quantum bit's superposition states wereseen between state “1” at v=0, i>0; “−1” state at v=0, i<0 and state “0”at v=0, i=0 at zero bias in FIG. 7 and FIG. 8.

EXAMPLE 7 Sensing ATP by Sensor 2 Using the CV Method and ATP PromotesSuper Conductivity

FIG. 5B depicts the Direct Electron Transfer (DET_(red)) peak currentincreased exponentially for Sensor 1 as the ATP concentration increasedfrom 40 fM to 60 nM compared with the control having 66.5, 146, 3048.7and 4664-fold increase in four levels, respectively at 60 Hz scan rate.FIG. 6 shows scan rate impacts on Sensor 2's i-V curves over scan ratefrom 1 Hz to 25 kHz in the Tris/HCl/MCD control media.

FIG. 7 shows Sensor 2's sine or cosine wave peak superconducting currentintensity increased or decreased exponentially for forwarding scan andbackward scan, respectively, over ATP concentrations from Panl B, 400aM; Panel C: ATP 400 fM; Panel D: ATP 200 pM; Panel E: ATP 200 nM; PanelF: ATP 2 μM. at zero-bias at the same scan rate 60 Hz compared with thecontrols in FIG. 6. We observed that a 25 aM ATP concentration is notenough to turn the memristive to superconductivity shown in Panel A. Thesuperposition characteristics were shown also as an example labeled inPanel F. The exponential increase of the superconducting current vs. ATPconcentrations over 400 aM to 2 μM for the forward scan against thebackward scan, which is in an exponential decay, were shown in FIG. 9.At high scan rate of 10 kHz, both the forward and the backward scan'ssupercurrent at zero-bias increased non-linearly from 200 fM till near 1μM, both current are dropped drastically as shown in FIG. 10. FIG. 10depicts the supercurrent vs. ATP concentration curves of the DET_(red)and DET_(ox) peaks at the zero-bias over ATP concentration ranges from200 fM to 200 μM. Data obtained according the figures from FIGS. 8A to8I.

EXAMPLE 9 ATP Induces Phase Change in Detail Presented in Sensor 2

Above Section we discovered ATP promoted superconductivity at anappropriate concentration and scan rate in the Tris buffer, now thisSection we explain the invention of the technology on Sensor 2discovered the ATP's another role which is for inducing a phase changeas shown in FIG. 11 for 10 consecutive scans at 60 Hz. The curves fromPanel A are with an ATP concentration 400 fM; Panel B with ATP 200 pM;Panel C with ATP 200 nM, and Panel D with ATP 2.0 μM, respectively. Thephases of the i-V curves were constantly changing at a fixed scan rate,but the ATP concentration were increased.

FIG. 12A depicts the i-V curves of a 25 aM low concentration ATP inconsecutive 5 scan cycles at a scan rate 60 Hz compared with the controlof Sensor 2. There was no phase change occur, and the hysteresis curvesare seen through the 5 consecutive scans. FIG. 12B depicts whenconcentration increased to 400 fM, in the consecutive 9 scan cycles ofthe same scan rate, we observed the cross-point moved away from origin,and the i-V curves having degree of superconductivity at zero-bias, andcompanied with a phase change occurred compared with the control. FIG.12C also depicts the similar trend when ATP concentration increased to200 nM ATP concentrations, Sensor 2 shows the phase changes andsuperconducting current.

EXAMPLE 10 The 3D Quantum Conducting Map in the Multiple-Variable Study

Quantum computing is computing using quantum-mechanical phenomena, suchas superposition and entanglement [28]. Superconducting flux qubit hastwo states that can be effectively separated from the other states isthe basic building block of quantum computers. Current DC or RFsuperconducting SQUID were made in advance for a faster switch time;however, hundreds of MHz electromagnetic field applied onto a tankcircuit coupled to the SQUID is needed for the system to work undercryogenic conditions [29-31]. The RF-SQUID consists of a superconductingring of inductance L interrupted by a JJ, the potential energy of theSQUID and the Hamiltonian equations are given by

U(Φ)=(Φ−Φ_(e))²/2L−E _(J) cos(2πΦ/Φ₀)   (3) [28]

H=Q ²/2C+(Φ−Φ_(e))²/2L−E _(J) cos(2πΦ/Φ₀)   (4) [28]

Φ_(e) is the applied magnetic flux penetrating the SQUID ring, Φ is thetotal magnetic flux threading the SQUID ring, L is the inductance, E_(J)represents the Josephson coupling energy, and Φ₀ is the superconductingmagnetic flux quantum, Q is the charge on junction's shunt capacitancesatisfying [Φ, Q]=ih/2π, while h is the Planck constant.

Stern's group reported the observation of Majorana bound states ofJosephson vortices in topological superconductors, and the equations ofthree types of energy contributions to the Josephson vortices in a longcircular junction in a Sine-Gordon system was published [32]. TheJosephson junction energy was from the Cooper pair, the magnetic energywas from the inductivity of the circular vortex, and the charge energywas from the SIS quantum capacitor-like device [32-33]. Our groupreported using a 3D dynamic map method, to elucidate themultiple-variables between the component energies contributing to thesuperconductivity of the vortex array system at room temperature withoutexternal magnetic field applied. Our experimental data were shown on thei-V curves and the AFM structure of the superlattice array. The modifiedSine-Gordon system energy for our d-wave vortex array is:

E ^(n) _(JJA)=(1/2)C ⁻¹ _(i)(Q−en _(1 . . . i))²   (5)

E ^(n) _(L)=(1/2)μ₀ N ² _(n=1..i) ·A·L ⁻¹ _(n=1..i) ·I ² _(n=1..i)   (6)

where E^(n) _(jjA) is the charge energy of Josephson Junction arrays atn=1..i; Q is the charge, C is the total capacitance at n=1..i, en is then quantum particles at 1..i data point with an energy periodic in h/efor Josephson effect for d-wave [34]; E^(n) _(L) is the Inductive energyinduced by the circular toroidal array. N is the turning number aroundthe toroidal porous at n=1..i, A is the cross-sectional area of theporous, L is the length of the wending, μ₀ is the magnetic permeabilityconstant in free space; I is current. The toroidal arrays are in seriesconnected. Recent publication regarding our FFTJJ multiple-variablestudy results in 3D dynamic maps was presented in the literature [34].In this invention, the multiple variables, such as the ATPconcentration, the applied potential affect on the quantum conductancewere studied through the 3D mapping method without decomposed thesuperconducting energy into several components.

FIG. 13 depicts the 3D dynamic map of the relationships over 5 level ATPconcentrations from 0.2 pM to 2.0 μM over the potential range between−40 mV to +40 mV to illustrate the relationship among ATPconcentrations, zero-bias potential and the differential quantumconductance using 10 kHz scan rate forwarding scan data. From the map,the quantum conductance values are correlating with the ATPconcentrations at zero-bias, except at higher concentration near 1.0 μM,it was dropped.

EXAMPLE 11 Quantitation of ATP in Biological Specimens TheChronoamperometric Method (CA) Measured by Sensor 1.

FIG. 14 depicts Sensor 1's current vs. time over ATP concentrations over100 aM to 400 nM compared with the control in pH 7.8 Tris/HCl/MCDsolution. FIG. 15 depicts the lower end concentration curves. Insertsare enlarged view for lower end concentration compared with controls.Inserts are the enlarged view of the curves at the lower concentrationcompared with the control. FIG. 16 in Panel A depicts the regressioncalibration curve of current density vs. ATP concentrations in alogarithm scale for the x-axe and the y-axe (9 levels, n=27 over 100 aMto 400 nM) with the regression equation (y log scale)Y=1.9+0.57*(logscale)χ, r=0.996, S_(Y/Ω)=0.16, p<0.0001. In inversion of the equation,0.57 is the power of the χ, and 1.9 is the intercept from the log-logplot. Hence the inversed equation is F(x)=79x^(0.57). FIG. 16 Panel Bdepicts Sensor 1's calibration curve over a linear range 400 fM to 170nM (7 levels, n=21) with the regression equation y=65.4+13.4x, r=0.997,Sy/x=65, p<0.0001. The Detection of Limits (DOL) of 0.56 fM over theanalytical range 100 aM-400 nM with a relative Pooled Standard Deviation(RPSD) value 0.9% (n=30).

EXAMPLE 12 The Open Circuit Potential Method (OPO)

Sensor 2's strong superconductivity has enabled the device to directreal-time monitor energy change under open circuit potential, under areagent-free, antibody-free condition. FIG. 17 depicts the voltagecurves exponentially increase as the ATP concentration increase comparedwith the control in the buffer media. FIG. 18 depicts the non-linearcalibration curve of voltage vs. concentrations over the range of 25 aMto 400 pM. FIG. 19 shows a plot for high-end ATP concentration withspontaneous voltage curves as the time (600 s) over 0.8 nM (a) to 2 μM(d). FIG. 20 shows the calibration curve over the higher ATPconcentration range from 0.8 nM to 2 μM.

EXAMPLE 13 The Double Step Chronopotentialmetry Method (DSCPO) by Sensor2

The ATP concentrations also can be detected in several seconds using theDSCPO method and setting the fixed current as ±10 nA, and each step 4 swith a data rate 1 kHz. FIG. 21 depicts Sensor 2's voltage vs. time(each step 4 s) curves by the Double-step Chronopotentialmetry (DSCPO)method in the presence of various ATP concentrations from 25 aM to 400nM in the Tris/HCl/MCD buffer compared with the control. Each sample runtriplicates. The curves show a positive correlation between the voltageintensity and the ATP concentrations in 7 levels compared with thelowest voltage from the control. FIG. 22A depicts the calibration curveof the normalized action potential divided by the mean signal vs. ATPconcentrations over 100 aM to 200 nM. The relative pooled standarddeviation (RPSD) is 0.24%. The linear semi-log plot gave an equation ofyscale(Y)=A+B*xscale(X) with A=0.37 and B=0.06, r=0.992, n=18,S_(y/logx)=0.027, p<0.0001. The DOL was found from this plot is 46 aM.FIG. 22B depicts the low end ATP's calibration curve from ATP 25 aM to400 aM in the Action potential results of the DER peaks after subtractedthe background signals with a RPSD error 1.3% with the signal increaserate of 0.016V/aM. The DOL result is 2 aM.

EXAMPLE 14 Accuracy and Imprecision

The USDA certified organic milk for infants was compared with human milk(Lee Biosolutions, MO) without prior sample preparation. Human milk wascollected from normal subjects who breastfeed 1 month-old newborn, eachsample run triplicates.

Methods validations were studied through the recovery experiments usingfresh human milk and USDA certified organic milk for infants as controlscompared with spiked 100 aM and 60 nM ATP and with or without low andhigh-level LPS challenges by the OPO method against each one's standardsand the controls. The accuracy recoveries were 94±0.14% and 91±0.16% at100 aM and 60 nM ATP for the human milk samples compared with organicmilk samples of 85±0.2% and 79%±0.2%, respectively using the OPO methodin traced back to the Tris standard control. The human mike control andthe organic milk control samples have an agreement related to theTris/HCl/MCD buffer sample controls (each type samples run triplicates)are 94.0±0.14% and 95.5±0.2%, respectively. The 5% difference isbelieved due to the artificial chaperone cage effect on the proteins ofthe milk, which is to lead the landscape free energy down to the lowestfor a right folding [29-30]. After corrected this effect, human milk'srecovery in the two levels' ATP challenges is 98±0.14% and 95±0.16%; theorganic milk recoveries are 90.5±0.2% and 84.5±0.2%, respectively. Thedata implies the chaperoning effect has more impact on organic milk thanthat of the human milk. In the two levels' LPS challenges (100 ag/mL, 60ng/mL), the recovery results are 96±0.16% and 105±0.14% vs. 105.5±0.28%and 95±0.19% for human milk and organic milk, respectively.

Point accuracy and imprecision was studied through the recoveryexperiments using spiked human milk and the USDA certified organic milksamples against the control samples with 2 levels of ATP concentrationsat 100 aM and 60 nM, respectively. We compared the measured results withthe calibration curve after subtraction of the voltage values fromcontrol samples using sensor 2 as shown in Table 1. We also studied theLPS effects on the recovery at 10 fg/mL and 90 fg/mL, respectively undera fixed ATP concentration of 60 nM. The results shown the recoveriesusing human milk and organic milk samples with the voltage method arehigher than 96% with an imprecision error less than or equals to 2% atthe 100 aM and 60 nM levels ATP compared with the controls, respectivelywithout LPS challenge. Using two levels of LPS challenges with the fixedATP 60 nM, the recoveries are 103±0.8%, 103±0.7% for human milk samplescompared with the spiked controls at the same level in the buffer; usingorganic milk samples, the recoveries are 97±1%, 18.8±0.3% at 10 fg/mLLPS and 90 fg/mL LPS, respectively traced back to the spiked controls atthe same level in the buffer. These results showed organic cow milksamples are vulnerable to the LPS attack in higher-level 90 fg/mL, whichcaused an unacceptable result in recovery, but the human milk samplesdemonstrated immunological advantage with 100% recovery with two levelsLPS challenges even under 60 nM ATP concentration.

The CA method was also used to access the accuracy and imprecision.Human milk control samples against the standard control samples in theTris/HCl/MCD buffer found no specimen interference having 101±6%traceable to the standards; In the presence of 30 fM ATP spiked in thehuman milk samples, i.e., the final ATP concentration is 30 fM in thesample, the recovery results are 100.0±7%; However, when 60 nM ATPpresents in the human milk samples, due to the immunological property,human milk samples eliminated all the ATP's effect, led to no signalswere measureable; In the presence of 30 fM ATP, under a 10 fg/mL LPSchallenge of the human milk samples, the recovery results are 100±10%;Under 30 fM ATP, using a 90 fg/mL LPS challenge, the human milk samplesproduce 3-fold high signal intensity,

For a comparison, the organic milk control samples were found having a21% of HSP 60-like chaperone interference for trace back to the standardcontrol samples with 79±2.6%; in the Tris/HCl/MCD buffer; Aftercorrected the interference, in the presence of 30 fM ATP spiked in theorganic milk samples, the recovery results are 100.0±4.4%; when 60 nMATP presents in the organic milk samples, the recovery results are102.3±0.02%; In the presence of 30 fM ATP, under a 10 fg/mL LPSchallenge of the organic samples, the recovery results are 49±0.2%;Using a 90 fg/mL LPS challenge to the organic milk samples, the recoveryresults are 130±3.6%.

EXAMPLE 15 Applications for Defining of the TransformationalImmunomodulant Between the Anti-inflammatory and the Pro-inflammatoryStatus

We primarily suggest the turning point of the ATP concentration fromanti-inflammatory to pro-inflammatory for a “healthy” BiomimeticMMP-2/HSP60 sensor 2 in the extracellular environment is higher than 800nM. FIG. 23 further shows a contour map relationship between the ATPconcentration (as the y-axis), and the applied potential (as the x-axis)and the quantum conductance (as Z-axis). It was observed that the rangebetween the highest quantum conductance values at zero-bias isassociated with the ATP concentration between 200 fM to 800 nM, so wedefine this range having the Physiological High-Frequency Oscillation(Phy-HFO) as the “Anti-inflammatory” range; when concentration higherthan 800 nM, the quantum conductance values are reduced, and this rangewas defined as the “Pro-inflammatory” in the extracellular ATPconcentration range at 10 kHz. Because Sensor 1 can be viewed as a“Mutated” or “Stressful” Biomimetic MMP-2/HSP60 model as far as thecapability to promoting the Cooper-pair electrons' concerns, was greatlydiminished as shown in the AFM image FIG. 2B, hence it was shown Sensor1 neither have superconducting peaks in the presence of ATP in thebuffer media in 60 Hz and 10 kHz scan rate, respectively.

EXAMPLE 16 Applications in Defining of ATP Concentration RangesEffecting on Cell Reversible Membrane Potential (RMP) and Its RatioBetween Action Potential and Resting Potential

It is a well-recognized phenomenon that cancer cells have abnormal cellmembrane potential [35-38]. Biologists measure cell membrane action andresting potentials with burdensome instrumentation and time-consumingprocedures. A recent report shows breast cancer cell division caused amembrane potential increase [38] due to variations in ion channelexpression. Because the normal cell membrane action potential is 58 mV,and −70 mV is for the resting potential [39], the small signals are veryeasily buried in the background noises [40] that can cause problems topediatric neurologist and intensive care unit doctors who need strongsignals to monitor and diagnose the neonatal neurological diseases [40].The consequences of human cancers, trauma brain injury (TBI) and otherdiseases are not be able to maintain mitochondrial cell's reversiblemembrane potential (RMP) and unable to maintain the normal membrane'spotential ratio between the action potential and the resting potentials[35-44]. Our group first used the biological marker of theaction/resting potential ratio to monitor the treatment oftriple-negative breast cancer and the brain cancer prognosis in a 3Dheat release map [41-44]. There is very few, if any, uses the ratio ofaction/resting potential as a biomarker to monitor and define the ATPconcentration ranges, that transforms from anti-inflammation topro-inflammation in the presence of bacterial toxins under awell-controlled system without any sample processing, no labeling and notracers were used. FIG. 24 shows the cell RMP voltage (after subtractingthe control) is not depending upon the ATP concentrations between therange from 100 aM to 800 nM with a power of 0.013, that means the cellvoltage increase rate is only 1.3% of the ATP concentration increaserate, hence this range is the “safe zone” confirmed by FIG. 10 using the3D map method based on the CV data. However, we found concentrationslower than 100 aM and higher than 800 nM the cell RMP can not keepconstant, either lower than the normal average cell voltage, or 10,20-fold higher than the normal average cell voltage, therefore, wedefine this range as the pro-inflammatory toxin range, for contrast, the“safe zone” range was defined as the anti-inflammatory toxin range.

FIG. 25 further shows the extracellular ATP concentration range effectson the ratio of the cell action potential vs. resting potential. Theresults showed the concentration lower than 100 aM or higher than 400 nMwill lead to a ratio of action vs. resting potential eithersignificantly lower or significantly higher than the normal ratio range,which is between 0.7 to around 1.0 [45-50]. Our prior works revealed theliving cancer cells can lead to an abnormal ratio up to 3, 10 to100-fold higher than this range according to the nature of cancer andthe stage of the cancers are in. Even FIG. 24 reveals the total cellvoltage at 800 nM is in the “safe zone” is respected to the energyconcerns, but clinically, the ratio value in FIG. 25 further revealedthe 800 nM ATP concentration led to a 10-fold higher than the normalratio range, hence in FIG. 25 we excluded the 800 nM points. The ratiovs. concentration curve has a power 0.08 that means the ratio increaserate is 8% of the concentration rate increase having a CV value±4.5%error in the normal range was demonstrated.

EXAMPLE 17 Applications in Assessing Human Milk Immunological Advantagein Preventing Extracellular ATP Hydrolyzation

Assessing human milk immunological advantage in preventing extracellularATP hydrolyzation is important. Our study was conducted through the CVmethod using the Sensor 1 because we knew Sensor 1 lacks Copper-pairelectrons, and we used the human milk samples compared with thecertified organic milk samples for infants under the same experimentalconditions. FIG. 26, Left Panel shows the extracellular hydrolysis DEToxcurves (as also called the memristive peak) happened in the presence ofspiked 60 nM ATP (final concentration), which caused a 9-fold increaseof the peak current at 700 mV compared with the organic milk negativecontrol. There is an unknown peak observed in the organic control milksample located at 20 mV, and the peak was the 2.8-fold increase of theamplitude when ATP presences. In contrast, FIG. 26 in the Right Panelshows the buffer control sample of the CV curve has no such unknown peakat 20 mV. There is a current increase for the DET_(ox) peak at 700 mVfor more than 173-fold in the presence of 60 nM ATP compared with thecontrol, and the DET_(red) peak intensity also increased 150-fold in thepresence of ATP compared with the control buffer sample. The DET_(ox)has a first-order ATP hydrolysis rate of 5.92×10⁻³/s by plotting thepeak currents vs. scan cycles (each cycle is 53.33 s) as shown in FIG.27. The top Panel curve is for the DET_(red) peak current vs. scancycles; the bottom Panel curve is for the DET_(ox) peak current vs. scancycles.

Human milk samples communicated with the “mutated” HSP60 membrane on thesame Sensor 1 having a different manner compared with the organic cowmilk samples. FIG. 28A, FIG. 28B, FIG. 28C and FIG. 28D depict the humanmilk samples under the impact of 60 nM ATP concentration in 4 scancycles (2 more cycles curves did not show) compared with the human milkcontrols using Sensor 1 at 60 Hz. The nodes, the super-positioning andthe phase change at the zero-bias were observed, and there was no ATPhydrolysis signature peak observed, and there was no unknown DETreduction peak noticed. FIG. 29 shows an alternative up and down peaksuperconducting current vs. scan cycles for the forward and backwardscan, respectively. The human milk samples have “eyes” that can see thedanger and purposely avoiding communicate with the ATP molecules becausein the human milk contains living microbiota, which the HSP60 and ZnTchaperonin proteins in the extracellular plays a role of the guardian'sprotection. FIG. 30 shows the overlapping curves of the control humanmilk sample in 6 consecutive scans vs. the milk sample with 60 nM ATP.The peak magnitude kept the same, but the phase change is constantlyhappening for with or without ATP, that indicates the human milkpromoting an orderly electromagnetic energy stored in the cell forenhancing the brain development and memory caused by the meminductivitythrough the Josephson junctions, in which is advantages compared withthe “chaos” electromagnetic energy acquired from Alzheimer's β-amyloidalaccumulation [51-54].

EXAMPLE 18 Experimental

Sensor 1 has an activated biomimetic MMP-2 membrane by a heating methodat 80° C. for 5 minutes using the innate biomimetic MMP-2 membranefabricated based on a published procedure [34]. Sensor 2 was also in astate of activation of biomimetic MMP-2 by a direct deposited methodwith compositions of TCD, PEG, PVP, bM-β-DMCD and embedded zinc chlorideon gold chips with appropriate proportions at 37° C. for 96 hours. TheUSDA certified organic milk for infants was compared with human milk(Lee Biosolutions, MO) without prior sample preparation. Human milk wascollected from normal subjects who breastfeed 1-month-old newborns, eachsample run triplicates.

The morphology of the AU/SAM was characterized using an Atomic ForceMicroscope (AFM) (model Dimension Edge AFM, Bruker, MA). Data collectedin TappingMode using silicon probes with a 5-10 nm tip radius and ˜300kHz resonance frequency (Probe mode TESPA-V2, Bruker, MA).

EXAMPLE 19 Conclusions and Discussions

A multiple functioning superconductive device was invented based onToroidal Josephson Junction (FFTJJ) array with 3D-cage structureself-assembled organo-metallic superlattice membrane. The device notonly mimics the structure and function of an activated MatrixMetalloproteinase-2 (MMP-2) protein, but also mimics the cylinderstructure of the Heat Shock Protein (HSP60) protein, that works at roomtemperature under a normal atmosphere, and without externalelectromagnetic power applied. The device enabled direct rapid real-timemonitoring atto-molarity concentration ATP in biological specimens andwas able to define the anti-inflammatory and pro-inflammatory statusrevealed a transitional range of ATP concentration under antibody-free,tracer-free and label-free conditions.

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What is claimed is:
 1. A multiple-functioning superconductive devicecomprising (a) an electrode has an organo-metallic superconductivemembrane having arrays of the 3D-nanocage structure by self-assembledcross-linked polymers with transition metal ions; (b) a directelectron-relay within a biomimetic matrix metalloproteinase-2 (MMP-2)membrane and a media comprising of imidazole modified cyclodextrin andATP formed chelating coordinating bounds, forming a long range directelectron-transfer (DET) chain; (c) the superconductive organo-metallicmembrane comprises of Toroidal Josephson Junction (FFTJJ) array. 2.According to claim 1, wherein the superconducting membrane hasFriedel-oscillation.
 3. According to claim 2, wherein theFriedel-oscillating superconductor mimics the cylinder structure of theHeat Shock Protein (HSP60) protein working at room temperature under anormal atmosphere, and without external electromagnetic power applied.5. According to claim 1, wherein the device direct rapid real-timemonitored atto-molarity concentration ATP in biological specimens havingpoint accuracy higher than 96% with imprecision errors less than orequals to 2% at a 100 aM and 60 nM levels ATP concentration levels,compared with the controls, respectively.
 4. According to claim 1,wherein the device has multiple-functioning of defining thetransformational immunomodulant between the Anti-inflammatory) and thePro-inflammatory status in extracellular ATP concentration range wasdemonstrated at 10 kHz.
 5. According to claim 1, wherein the device hasmultiple-functioning of monitoring the normality of the cell reversiblemembrane potential (RMP) for the ATP concentration between 100 aM to 800nM using the voltage method at ±10 nA with each potential step at 0.25Hz.
 6. According to claim 1, wherein the device has multiple-functioningof monitoring the clinical normality of the ratio of the cell actionpotential vs. resting potential in ATP extracellular level higher than100 aM and lower than 400 nM is normal in the ratio.
 7. According toclaim 1, wherein the device has multiple-functioning used for assessingmilk immunological characteristics in preventing extracellular ATPhydrolyzation using biological specimens.
 8. According to claim 1,wherein the biomimetic activated MMP-2 superconductive device (throughheating for activation) direct quantitatively detect ATP in the rangebetween 100 aM to 200 nM with a relative pooled standard deviation(RPSD) 0.24%, and a DOL value is 46 aM using the voltage method.